Recent studies suggest that positive selection is driving rapid divergence of genes involved in reproductive isolation between species. Detailed characterization of reproductive isolation genes will therefore contribute to our knowledge both of species-specific traits and adaptations and of the speciation process itself. We have) recently shown that the gene Hmr causes Fl hybrid lethality between Drosophila melanogaster and its sibling; species, and suggested that a second gene, Lhr, is also involved in this lethality. In Aim 1 of this proposal we propose to clone the Lhr gene in order to test the hypothesis that hybrid lethality is caused by the interaction of the D. melanogaster Hmr gene and the sibling-species Lhr gene. We will attempt to reconstitute the interspecific incompatibility entirely within /). melanogaster strains using transgenic constructs. We will also create a loss-of-function mutation in Lhr in D. melanogaster in order to understand the function of Lhr within species. Aim 2 will explore the relationship between the molecular evolution and the functional properties ct Hmr with respect to its intraspecific and hybrid functions. We will do developmental analyses of Hmr loss of- function mutants and of interspecific hybrids, use molecular evolutionary and population genetic techniques to identify regions and codons of Hmr that may be diverging under selection, and then use transgenic assays to test the functional consequences of hypotheses derived from these evolutionary analyses. We will also investigate whether Lhr has diverged under selection between D. melanogaster and its sibling species. In Aim 3 a genetic screen will be done in D. melanogaster to identify new genes that interact with Hmr and Lhr to cause hybrid lethality. The forthcoming whole-genome sequence from the sibling species D. simulans as well as several other Drosophila species will greatly enhance both the technical feasibility and the potential impact of this proposal as a model for testing functional consequences of sequence divergence.